This invention relates to an electrophoresis cassette and comb with filling means which enables the cassette with comb in place to be filled with the liquid electrophoretic medium. The cassette is useful for conducting gel electrophoresis. The invention also relates to a method for filling electrophoresis cassettes with the comb in place.
Electrophoresis is the resolution of a complex mixture of macromolecules on the basis of charge and/or size under the influence of an electric field. Usually, separation of the charged molecules is based on the strength of the electrical field and the net charge, size and shape of the molecules. The separation can also, at least in part, be affected by other parameters, such as isoelectric points, ionic strength, viscosity and temperature of the medium in which the charged molecules are moving. Since proteins and other biological molecules, such as DNA, RNA, enzymes, carbohydrates and the like are charged, electrophoresis techniques are ideal to separate them for analytical or preparative purposes.
It is common practice to use a cassette for conducting gel electrophoresis. The cassette comprises two flat plates, usually transparent glass or plastic, separated by spacing means and sealed along the sides and bottom edges to provide a void or confined space between the plates for placement of a suitable separation gel medium or electrophoresis gel such as agarose or a polyacrylamide in liquid form and its subsequent gelling or polymerization. When a separation is run on the gel in the cassette, the top portion of the gel is in contact with a buffered solution and the lower portion of the gel is in contact with a second buffered solution. An electric current is applied to the buffered solution(s) or the gel causing the migration and separation of the samples. By convention, the samples, for example, negatively charged proteins and nucleic acids, tend to run from the cathode (the top of the cassette, where the sample is placed) to the anode of the gel.
For the purposes of describing this invention, reference to the top of a cassette means the end of the cassette at which the wells are formed and in the instance of negatively charged samples, the cathode end of the gel. Additionally, the use of the terms "gelled" and "gel" refers to a solid state of the electrophoretic medium whether it be due to the cooling of an agarose gel or the polymerization of an acrylamide or acrylamide derivative.
In protein or nucleic acid fragment analysis, it is desirable to run more than one sample in a gel and to keep the samples separate. This is accomplished by sample separating means which is most commonly provided by the formation of wells at the sample-loading end of the gel. Sample wells are formed usually by means of a removable piece or "comb" with teeth having the desired shape of the wells and having straight (level) substantially perpendicular ends to the teeth. The comb is brought into contact with the liquid separation medium before it gels or polymerizes and is positioned so that the teeth extend into the liquid medium while it gels. After the gel has set, the comb is removed to leave wells where samples can be positioned. When conducting nucleic acid sequencing it is desirable to use a spacer, i.e., a comb with one tooth extending substantially across the width of the void or confined space, to create a straight edge to the resulting gel which edge is substantially perpendicular to the front and back plates of the cassette. Upon completion of the gelation of the electrophoretic medium, the comb (spacer) is removed and usually a saw-tooth configured (sharks-tooth) sample separator is inserted into the top of the cassette with the tip of the teeth touching or entering into the gel surface and the body of the teeth forming a reservoir for the samples to be analyzed. The samples enter the gel between the tips of the teeth. Often the sharks-tooth sample well configuration is the opposite side of the spacer. The term "comb" used to describe this invention, unless the context indicates otherwise, refers either to having one-tooth or -finger (a spacer) or a plurality of teeth or fingers.
To improve the separation of certain types of molecules, it is common practice to change the composition of the gel medium in the direction of the electrophoresis. This change in composition can, for example, take the form of a discontinuity formed by a stacking gel at the upper most portion of the gel and a resolving gel at the lower most portion of the gel. Another example of a change in composition is the commonly used gradient gel consisting of a continuous gradient or a discreet gradient of bands each of uniform but different composition or any combination of continuous and discreet gradient. In order to provide accurate sample resolution, it is necessary that the desired electrophoresis composition remain undisturbed until the gel medium has gelled or polymerized completely, particularly in the instance of stacking and/or gradient gels.
There are two commonly used methods for introducing the liquid separation medium into the cassette prior to polymerization, each with its own distinct advantages and disadvantages. Put simply, the solution can be introduced from the top of the cassette or from the bottom of the cassette.
When filled from the bottom of the cassette, the main advantage is that the comb can be in-place during the fill and the potential disruption during insertion of the comb of any desirable gradations or discontinuities to the liquid separation medium during filling is avoided. A disadvantage of the bottom fill is the need to gain access to the bottom of the cassette to introduce the gel medium. This access to the bottom of the cassette must be sealed until the liquid medium has gelled or leaking of the ungelled material will result. One attempt to minimize the disadvantage of the bottom fill method is exemplified by the commercially available gel casting apparatus by Hoefer Scientific called SE 275 Mighty Small Four-Gel Caster. Rather than effecting a seal with an opening in the bottom of a cassette, the Hoefer apparatus submerges an open bottomed cassette in a tub of liquid medium. Some of the liquid medium finds its way into the cassette. The cassette is left submerged in the tub of liquid medium until it has gelled at which time the cassette can be removed from the tub, cleaned up and used. This method, although producing a usable filled cassette, has the added disadvantage of wasting the polymerized gel medium that did not go into a cassette.
When filled from the top of the cassette, the main advantage is that the cassette can be filled without concern for sealing an opening until after the gel medium has polymerized. A disadvantage of the top fill is the requirement that the comb be placed into the cassette and into contact with the gel medium after the cassette is filled and thereby greatly increasing the risk of disturbing the gel during its formation, particularly any desirable gradations or discontinuities introduced into the gel medium during filling.
Although the above mentioned disadvantages for the bottom fill method and top fill method are real when making gels one at a time in the laboratory, they can be addressed to some extent by skill and careful attention to detail. It is when gels are made on a production scale that the disadvantages become pronounced. When using the bottom fill method, it is not easy to keep the bottom sealed during the gellation or polymerization step which can take as long as an hour. Any leakage not only ruins the cassette that is leaking, but also can create a mess on the production line. When a submerge-fill method is used, the attendant waste of gel medium and requirement to clean the cassettes are less than desirable.
Additionally, the uniformity of the gel is important to the quality of the separation obtained, for example, uniformity in the porosity of the gel or regions of the gel, uniformity (level and perpendicular to the plates) in the edge of the gel which first contacts the samples, uniformity in the thickness of the gel and uniformity (level and perpendicular to the plates) in the interface between discreet bands of gels (for example, between gradient gels or between stacking and resolving gels). Thus, it is desirable that the liquid gel-forming medium introduced into the cassette be disturbed as little as possible while gelling.
Accordingly, the present invention provides a cassette filling means for a cassette for gel electrophoresis that retains the advantages of the bottom fill method and the top fill method without the attendant disadvantages.